Charles W. Hewitt

COMPOSITE TISSUE (LIMB) ALLOGRAFTS IN RATS: II. INDEFINITE SURVIVAL USING LOW-DOSE CYCLOSPORINE

Cyclosporine has reawakened interest in transplantation of peripheral composite tissue allografts (CTA) of skin, muscle, bone, vessel, and nerves. The purpose of this study was to examine whether cyclosporine could produce indefinite survival of CTA. Two groups of LEW recipients of LBN limb transplants were given different long-term treatments of cyclosporine. Tolerance was achieved in many of the animals. Several possibilities for the mechanism of this tolerance are discussed.

Development of stable mixed T cell chimerism and transplantation tolerance without immune modulation in recipients of vascularized bone marrow allografts

A consistent majority (62.5%) of immunologically unmodified rat recipients transplanted with vascularized hind-limb bone marrow allografts across a semiallogeneic transplant barrier developed tolerance with absence of graft-versus-host disease. A minority of recipients (37.5%) demonstrated lethal GVHD. Transplantation tolerance in the majority was associated with the induction of stable low-level mixed T cell chimerism, including donor CD5+, CD4+, and CD8+ lymphocytes. Chimeras were specifically immune nonresponsive to host alloantigenic determinants. These results emphasized a potentially important mechanism for low-level stable mixed lymphoid chimerism (SMLC) in tolerance induction, independent of immune suppressive effects due to irradiation or immunopharmacologic intervention. These vascularized bone marrow transplantation (VBMT) results may establish the experimental foundation for a novel approach to stem cell transfer and bone marrow transplantation.

Composite tissue (limb) allografts in rats. III: Development of donor-host lymphoid chimeras in long-term survivors

Eight LEW rat recipients possessing long-term-surviving (206-701 days) LBN vascularized hind limb allografts (CTAs) were tested for donor-host lymphoid chimerism. The recipients received various cyclosporine (CsA) treatment protocols in order to induce indefinite CTA acceptance. Histological examination of long-term-surviving CTAs demonstrated normal-appearing bone marrow in the donor limb. Lymphocytes isolated from host hemopoietic tissues (peripheral blood and/or spleen) by ficoll-hypaque density gradient centrifugation were tested against LEW-anti-BN antisera. Comparisons were made to standard curves employing various known concentrations of LBN and LEW cell combinations. The level of lymphocyte agglutination (dependent variable) showed a significant (P less than 0.025-0.005) linear relationship to the concentration of LBN donor cells (independent variable) present. Lymphocyte suspensions isolated from long-term CTA host peripheral blood and/or spleen showed a mean of 19.7% (+/- 9.7-95% confidence interval) donor LBN mononuclear cells present. Thus, it appeared that lymphoid cells originated from, and/or were released from LBN donor bone marrow into the circulation, resulting in chimeric repopulation of hemopoietic tissues. The presence of donor immunocytes in these limb allograft recipients may have been beneficial, and thus could have helped contribute to the long-term CTA survival observed.

Composite tissue (limb) allografts in rats. I: Dose-dependent increase in survival with cyclosporine

The dose-response effect of cyclosporine on rat limb transplant prolongation was investigated across the LBN-to-LEW histocompatibility barrier. This composite tissue allograft model has been shown to represent a strong transplantation barrier. Median limb allograft survival times increased in a dose-dependent manner with low cyclosporine doses, and then reached a plateau at higher levels. The cyclosporine dose that produced half-maximal survival based on a 20-day treatment was only 3.7 mg/kg/day. Histopathology revealed that the rejection process was distinctly different in limb allografts treated with cyclosporine compared with noncyclosporine-treated controls. Rejection appeared to be delayed or partly arrested in certain areas of cyclosporine-treated limb allografts. These studies represent an initial step in laying the experimental foundation for clinical transplantation of composite tissue allografts using cyclosporine-induced immune suppression.

The Effects of Testicular Trauma on Fertility in the Lewis Rat and Comparisons to Isoimmunized Recipients of Syngeneic Sperm

Adult male Lewis (LEW) rats were used to investigate the effects of unilateral testicular trauma on fertility. Comparisons were made between normal and experimental rats immunized with syngeneic sperm in Complete Freunds Adjuvant (CFA). Matings within the three groups yielded offspring to all normal males, no offspring to the immunized rats, and 27% (3/11) fertility in the trauma group (p less than 0.001). The contralateral testis demonstrated decreased volumes, various degrees of aspermatogenesis and smaller seminiferous tubular diameters, in both the trauma and immunized groups compared to the controls. Similar histopathologic findings of chronic granulomatous inflammation within contralateral testes in both the trauma and immunized groups suggested a common immune etiology for infertility via possible disruption of the blood-testis barrier.

An extraperitoneal isolated vascularized bone marrow transplant model in the rat.

An isolated vascularized bone marrow transplant (iVBMT) model was previously developed in the rat to specifically study the role of bone marrow and its environment in a composite tissue allotransplant. An extraperitoneal model was successfully created to avoid laparotomy and cross-clamping of the great vessels. The extraperitoneal iVBMT model consisted of a left donor femur that was harvested with its nutrient vessels, anastomosed to the right femoral vessels in a syngeneic host, and then placed subcutaneously in the abdominal wall. At explant, the graft vessels were grossly patent, and histology of the graft bones showed a viable marrow compartment. Polymerase chain reaction demonstrated peripheral chimerism in the recipients. This model is technically simple with minimal morbidity in the recipient animals. By using the iVBMT, future studies across semiallogeneic and allogeneic barriers will help define the role of the bone marrow compartment in composite tissue allotransplants to potentially induce immune tolerance.

A rapid and sensitive cellular enzyme-linked immunoabsorbent assay (CELISA) for the detection and quantitation of antibodies against cell surface determinants. I. A comparison of cell fixation and storage techniques

A solid phase cellular ELISA was designed and evaluated for the detection of antibodies specific for cell surface determinants. It was hypothesized that certain fixation and freezing procedures would result in stabilization of cell structures for prevention of antigen diffusion and extraction during washing procedures. This would assure assay accuracy and convenient sample management. It was hypothesized that fixation with certain reagents prior to analysis would not alter antigenicity of antibody targeted epitopes. In order to improve the preservation of the cells following cell binding to the solid phase matrix while still retaining antigenicity and morphology, a series of fixatives and storage procedures were screened to determine which were best suited for CELISA. Methanol, washing buffer (WB), Hanks balanced salt solution (HBSS), and 0.5% formalin in HBSS were examined by comparing their relative cell binding capacity and the subsequent cell morphology. In consideration of all variables, fixation in 0.5% formalin provided the best maintenance of cell antigenicity, morphology, binding, and was associated with consistent results. Cells used immediately after fixation and fixed cells used after storage at -80 degrees C for up to 12 months were compared to determine if long term storage affected antigenicity. Since frozen cells and fresh cells demonstrated statistically identical positive to negative ratios and consistency of antibody binding, it was determined that long term frozen storage of formalin-fixed cells did not adversely affect antibody binding capacity to cell surface determinants.

Absence of graft-versus-host disease in the isolated vascularized bone marrow transplant.

An isolated vascularized bone marrow transplant (iVBMT) model was developed to study the contribution of the bone marrow component in a composite tissue allograft. We hypothesized that the iVBMT would be functional and cause graft-versus-host disease (GVHD) in a fraction of the recipients. Lewis iVBMT grafts were transplanted to Lewis-Brown Norway recipients. Animals were sacrificed at various times from 1 to 14 weeks. Polymerase chain reaction for microchimerism was performed on the hosts marrow. No animals exhibited signs of GVHD at death. Histologic examination of the grafts showed a normal mix of hematopoietic and fatty elements and appeared to be functional. Tissues usually affected-tongue, ear, liver, and gut-also showed no evidence of disease. Polymerase chain reaction demonstrated microchimerism in both groups. These findings suggest that the vascularized bone marrow within a composite tissue allograft is not the component that causes GVHD; rather, it may serve an immunomodulatory function for tolerance induction.

Cyclosporine and skin allografts for the treatment of thermal injury. I. Extensive graft survival with low-level long-term administration and prolongation in a rat burn model.

The hypothesis tested in the present and accompanying study is that and effective treatment for severe burns involves early excision of necrotic tissute followed by skin allografting ad cylosporine (CsA) immunosuppressive therapy. LEW (RR11) rats served as recipients of thermal injury and/or skin allografts. BNxLEW F1M (LBN, RT11+n) rats served as skin donors. LEW burn recipients received a hot water (90oC for 10 sec) 30% body surface area (BSA) full-thickness burn. As expected, LEW recipients treated with CsA (25 mg/kg/day for 20 days) demonstrated singnificant graft polongation compared with controls (P<0.005). Skin graft survival was similarly prolonged in LEW recipeints undergoing burn unjury, primary wound excision, and CsA administration comkpared with not increased in the thermal injury—CsA-treated recipients comkpared with burn controls. A final experiment was initiated to investigate how low-level long-term (>100 days) maintenace CsA treatment influenced skin allograft survivalfor possible future consideration in burn trauma. Recipioents receiving skin allografts plus CsA (20 days, 8mg/kg/day, followed by every other day therafter) did not reject their grafts. However, a possible early sign of rejection ( a gingle small ulceratiove lesion) was noted in five of these long-term CsA-treated animals at a meanof 3411 (SD) days. The lesion in these animals did not progress any further during CsA administration. His-topathologic study of selected animals removed from the CsA maintenance regimen for greter than 50 days following long-term administration revealed a number of interesting chronic lesions similar to thosse previously reported in the skin comonent of composite tissue (limb) allografts following long-term low-level CsA intervention. In conclusion, CsA was very successful in preventing rejection of skin allografts in a rat burn model without apparent adverse effects.

Reconstructive Allotransplantation: Considerations Regarding Integumentary/Musculoskeletal Grafts, Cyclosporine, Wound Coverage in Thermal Injury, and the Immune Response

With the advent of cyclosporine, a powerful and selective immunosuppressant, comes resurgence of a long-sought goal: to transplant modules of allointegumentary/musculoskeletal tissues or components thereof for the repair of peripheral tissue defects. Because these modules of integumentary and/or musculoskeletal tissue are actually composites of various tissues, they are also known as composite tissue allografts. The immediate goal of the studies reviewed herein is to lay the foundation in transplant immunobiology for the clinical exploitation of composite tissue allografts. The objective of these continuing studies is to induce permanent acceptance of composite tissue allografts. The value of such grafts lies in their potential for complete functional and cosmetic restoration in the surgical reconstruction of tissue after full-thickness burn injury. The initial results of basic experiments with cyclosporine are extremely encouraging in regard to the clinical potential for integumentary/musculoskeletal grafts in reconstructive allotransplantation.